Mini-HA Is Superior to Full Length Hemagglutinin Immunization in Inducing Stem-Specific Antibodies and Protection Against Group 1 Influenza Virus Challenges in Mice.

Seasonal influenza vaccines are updated almost annually to match the antigenic drift in influenza hemagglutinin (HA) surface glycoprotein. A new HA stem-based antigen, the so-called "mini-HA," was recently shown to induce cross-protective antibodies. However, cross-reactive antibodies targeting the HA stem can also be found in mice and humans after administration of seasonal vaccine. This has raised the question whether in similar conditions such a mini-HA would be able to show an increased breadth of protection over immunization with full length (FL) HA. We show in mice that in a direct comparison to H1 FL HA, using the same immunization regimen, dosing and adjuvant, a group 1 mini-HA has a higher protective efficacy against group 1 influenza virus challenges not homologous to the H1 FL HA. Although both antigens induce a similar breadth of HA subtype binding, mini-HA immunization induces significantly more HA stem-specific antibodies correlating with survival. In addition, both mini-HA and H1 FL HA immunization induce influenza neutralizing antibodies while mini-HA induces significantly higher levels of mFcγRIII activation, involved in Fc-mediated antibody effector functions. In agreement with previous findings, this confirms that more than one mechanism contributes to protection against influenza. Together our results further warrant the development of a universal influenza vaccine based on the HA stem region.

Mini-hemagglutinin vaccination induces cross-reactive antibodies in pre-exposed NHP that protect mice against lethal influenza challenge.

Seasonal vaccines are currently the most effective countermeasure against influenza. However, seasonal vaccines are only effective against strains closely related to the influenza strains contained in the vaccine. Recently a new hemagglutinin (HA) stem-based antigen, the so-called "mini-HA", has been shown to induce a cross-protective immune response in influenza-naive mice and non-human primates (NHP). However, prior exposure to influenza can have a profound effect on the immune response to subsequent influenza infection and the protective efficacy of vaccination. Here we show that mini-HA, compared to a trivalent influenza vaccine (TIV), elicits a broadened influenza-specific humoral immune response in NHP previously exposed to influenza. Serum transfer experiments showed that antibodies induced by both mini-HA and seasonal vaccine protected mice against lethal challenge with a H1N1 influenza strain heterologous to the H1 HA included in the TIV. However, antibodies elicited by mini-HA showed an additional benefit of protecting mice against lethal heterosubtypic H5N1 influenza challenge, associated with H5 HA-specific functional antibodies.

The activation energy for insertion of transmembrane alpha-helices is dependent on membrane composition.

The physical mechanisms that govern the folding and assembly of integral membrane proteins are poorly understood. It appears that certain properties of the lipid bilayer affect membrane protein folding in vitro, either by modulating helix insertion or packing. In order to begin to understand the origin of this effect, we investigate the effect of lipid forces on the insertion of a transmembrane alpha-helix using a water-soluble, alanine-based peptide, KKAAAIAAAAAIAAWAAIAAAKKKK-amide. This peptide binds to preformed 1,2-dioleoyl-l-alpha-phosphatidylcholine (DOPC) vesicles at neutral pH, but spontaneous transmembrane helix insertion directly from the aqueous phase only occurs at high pH when the Lys residues are de-protonated. These results suggest that the translocation of charge is a major determinant of the activation energy for insertion. Time-resolved measurements of the insertion process at high pH indicate biphasic kinetics with time constants of ca 30 and 430 seconds. The slower phase seems to correlate with formation of a predominantly transmembrane alpha-helical conformation, as determined from the transfer of the tryptophan residue to the hydrocarbon region of the membrane. Temperature-dependent measurements showed that insertion can proceed only above a certain threshold temperature and that the Arrhenius activation energy is of the order of 90 kJ mol(-1). The kinetics, threshold temperature and the activation energy change with the mole fraction of 1,2-dioleoyl-l-alpha-phosphatidylethanolamine (DOPE) introduced into the DOPC membrane. The activation energy increases with increasing DOPE content, which could reflect the fact that this lipid drives the bilayer towards a non-bilayer transition and increases the lateral pressure in the lipid chain region. This suggests that folding events involving the insertion of helical segments across the bilayer can be controlled by lipid forces.

Folding kinetics of an alpha helical membrane protein in phospholipid bilayer vesicles.

We report a detailed kinetic study of the folding of an alpha-helical membrane protein in a lipid bilayer environment. SDS denatured bacteriorhodopsin was folded directly into phosphatidylcholine lipid vesicles by stopped-flow mixing. The folding kinetics were monitored with millisecond time resolution by time-resolving changes in protein fluorescence as well as in the absorption of the retinal chromophore. The kinetics were similar to those previously reported for folding bacteriorhodopsin in detergent or lipid micelles, except for the presence of an additional apoprotein intermediate. We suggest this intermediate is a result of the greater internal two-dimensional pressure present in these lipid vesicles as compared to micelles. These results lay the groundwork for future studies aimed at understanding the mechanistic origin of the effect of lipid bilayer properties on protein folding. Furthermore, the use of biologically relevant phosphatidylcholine lipids, together with a straightforward rapid mixing process to initiate the folding reaction, means the method is generally applicable, and thus paves the way for an improved understanding of the in vitro folding of transmembrane alpha-helical proteins.

Controlling the folding efficiency of an integral membrane protein.

Research into the folding mechanisms of integral membrane proteins lags far behind that of water-soluble proteins, to the extent that the term protein folding is synonymous with water-soluble proteins. Hydrophobic membrane proteins, and particularly those with transmembrane alpha-helical motifs, are frequently considered too difficult to work with. We show that the stored curvature elastic stress of lipid bilayers can be used to guide the design of efficient folding systems for these integral membrane proteins. The curvature elastic stress of synthetic phosphatidylcholine/phosphatidylethanolamine lipid bilayers can be used to control both the rate of folding and the yield of folded protein. The use of a physical bilayer property generalises this approach beyond the particular chemistry of the lipids involved.

A stable trimeric influenza hemagglutinin stem as a broadly protective immunogen.

The identification of human broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin (HA) stem revitalized hopes of developing a universal influenza vaccine. Using a rational design and library approach, we engineered stable HA stem antigens ("mini-HAs") based on an H1 subtype sequence. Our most advanced candidate exhibits structural and bnAb binding properties comparable to those of full-length HA, completely protects mice in lethal heterologous and heterosubtypic challenge models, and reduces fever after sublethal challenge in cynomolgus monkeys. Antibodies elicited by this mini-HA in mice and nonhuman primates bound a wide range of HAs, competed with human bnAbs for HA stem binding, neutralized H5N1 viruses, and mediated antibody-dependent effector activity. These results represent a proof of concept for the design of HA stem mimics that elicit bnAbs against influenza A group 1 viruses.

A biological porin engineered into a molecular, nanofluidic diode.

We changed the nonrectifying biological porin OmpF into a nanofluidic diode. To that end, we engineered a pore that possesses two spatially separated selectivity filters of opposite charge where either cations or anions accumulate. The observed current inhibition under applied reverse bias voltage reflects, we believe, the creation of a zone depleted of charge carriers, in a sense very similar to what happens at the np junction of a semiconductor device.

Chemical modification of the bacterial porin OmpF: gain of selectivity by volume reduction.

OmpF is an essentially nonselective porin isolated from the outer membrane of Escherichia coli. Here we report on the manipulation of the ion selectivity of OmpF by chemical modification with MTS reagents (MTSET, MTSEA, and MTSES) and the (rather bulky) tripeptide glutathione, all cysteine specific. When recorded in a gradient of 0.1//1 M CaCl2 or 0.1//1 M NaCl, pH 7.4 solutions, measured reversal potentials of the most cation-selective modified mutants were (virtually) identical to the Nernst potential of Ca2+ or Na+. Compared to this full cation selectivity, the anion-selective modified mutants performed somewhat less but nevertheless showed high anion selectivity. We conclude that a low permanent charge in combination with a narrow pore can render the same selectivity as a highly charged but wider pore. These results favor the view that both the electrostatic potential arising form the fixed charge in the pore and the space available at the selectivity filter contribute to the charge selection (i.e., cation versus anion selectivity) of a biological ion channel.

Conductance and selectivity fluctuations in D127 mutants of the bacterial porin OmpF.

A recent molecular dynamics study questioned the protonation state and physiological role of aspartate 127 (D127) of E. coli porin OmpF. To address that question we isolated two OmpF mutants with D127 either neutralized (D127N) or replaced by a positively charged lysine (D127K). The charge state of the residue at position 127 has clear effects on both conductance and selectivity. The D127K but not the D127N mutant expresses resilient conductance and selectivity fluctuations. These fluctuations reflect, we think, either changes in the ionization state of K127 and/or transitions between unstable subconformations as induced by the electrostatic repulsion between two positively charged residues, K127 and the nearby R167. Our results slightly favor the view that in WT OmpF residue D127 is deprotonated. As for the role of D127 in OmpF functionality, the gating of both mutants shows very similar sensitivity toward voltage as WT OmpF. Moreover, the current fluctuations of the D127K mutant were observed also in the absence of an applied electric field. We therefore dismiss D127 as a key residue in the control mechanism of the voltage-dependent gating of OmpF.

Ca2+ selectivity of a chemically modified OmpF with reduced pore volume.

We studied an E. coli OmpF mutant (LECE) containing both an EEEE-like locus, typical of Ca(2+) channels, and an accessible and reactive cysteine. After chemical modification with the cysteine-specific, negatively charged (-1e) reagents MTSES or glutathione, this LECE mutant was tested for Ca(2+) versus alkali metal selectivity. Selectivity was measured by conductance and zero-current potential. Conductance measurements showed that glutathione-modified LECE had reduced conductance at Ca(2+) mole fractions <10(-3). MTSES-modified LECE did not. Apparently, the LECE protein is (somehow) a better Ca(2+) chelator after modification with the larger glutathione. Zero-current potential measurements revealed a Ca(2+) versus monovalent cation selectivity that was highest in the presence of Li(+) and lowest in the presence of Cs(+). Our data clearly show that after the binding of Ca(2+) the LECE pore (even with the bulky glutathione present) is spacious enough to allow monovalent cations to pass. Theoretical computations based on density functional theory combined with Poisson-Nernst-Planck theory and a reduced pore model suggest a functional separation of ionic pathways in the pore, one that is specific for small and highly charged ions, and one that accepts preferentially large ions, such as Cs(+).

A light-actuated nanovalve derived from a channel protein.

Toward the realization of nanoscale device control, we report a molecular valve embedded in a membrane that can be opened by illumination with long-wavelength ultraviolet (366 nanometers) light and then resealed by visible irradiation. The valve consists of a channel protein, the mechanosensitive channel of large conductance (MscL) from Escherichia coli, modified by attachment of synthetic compounds that undergo light-induced charge separation to reversibly open and close a 3-nanometer pore. The system is compatible with a classical encapsulation system, the liposome, and external photochemical control over transport through the channel is achieved.

Permeation properties of an engineered bacterial OmpF porin containing the EEEE-locus of Ca2+ channels.

The selectivity filter of the bacterial porin OmpF carries a small net charge close to -1 e and is therefore only slightly cation-selective. Calcium channels, on the other hand, contain four negatively charged glutamates, the EEEE-locus, and are among the most selective cation channels known. We aimed to turn the essentially nonselective OmpF into a Ca2+-selective channel. To that end, two additional glutamates (R42E and R132E) were introduced in the OmpF constriction zone that already contains D113 and E117. Mutant OmpF containing this DEEE-locus has a high Ca2+ over Cl- selectivity and a Na+ current with a strongly increased sensitivity to 1 mM Ca2+. The charge/space competition model, initially applied to the L-type Ca2+ channel, identifies the fixed charge and filter volume as key determinants of ion selectivity, with the precise atomic arrangement having only second-order effects. By implication, the reproduction of fixed charge and filter volume should transform two channels into channels of similar selectivity, even if the two belong to entirely different ion channel families, as is the case for OmpF and the L-type Ca2+ channel. The results presented here fit quite well in the framework of charge/space competition theory.